Lab Practical Flashcards
(122 cards)
1
Q
TSA plate
A
- trypticase soy agar
- supplies nutrients for many types of bacteria and fungi to grow
- in a petri plate
2
Q
NB Tube
A
- nutrient broth
- contains nutrients supporting growth of organisms in a liquid medium
3
Q
Brightfield Microscopy
A
-for stained cells
4
Q
Phase Microscopy
A
- for living cells
- to examine motility
5
Q
Telescopic Eyepiece
A
-used to align annulus diaphragm and phase ring
6
Q
NA Slant
A
- nutrient agar slant
- agar cooled on a slant so it is an angle in the tube
7
Q
Streak Plate
A
- make dot
- fan out 5-6 lines from dot, flame loop
- fan out 5-6 lines from ends of last lines, flame loop
- do this two more times so you have four sets of streaks
- used to isolate pure colonies
8
Q
Bacterial Morphology
A
- rod
- coccus
- spiral
9
Q
Coccus
A
- staphylococcal: cocci in clusters
- streptococcal: cocci in chains
- tetrad: four
- diplococcus: pairs
10
Q
Types of Flagellation
A
- polar: one at one or both ends
- lophotrichous: tuft at one end
- peritrichous: all around cell
- amphitrichous: tuft at both ends
11
Q
Types of bacterial movement
A
- true motility: random movement in all directions
- brownian: bombarded on all sides by water molecules
- streaming: caught in convection currents; all moving in same direction
12
Q
Wet Mount
A
- drop of water and one loopful of organism
- use phase contrast
- too much light means can’t see
13
Q
Hanging Drop
A
- concave slide
- used to study motility
- 2 loopfuls on coverglass then use vaseline to fasten to depression slide
14
Q
Motility Test
A
- semisolid media .3 or .4% vs 1.5% agar which allows motile bacteria to move through media
- contains tetrazolium dye that turns red where there is growth
- semisolid or SIM (sulfur indole motility) media
- proteus displays true motility with this test
- positive tubes have red radiating out from the stab
15
Q
Simple Staining
A
- use of a single stain to color a bacterial cell
- common are crystal violet, methylene blue, and basic fuchsin which are basic dyes
- used to determine morphology of cells
16
Q
Pleomorphism
A
-irregularity of form; demonstrating several different shapes
17
Q
Metachromatic Granules
A
-distinct reddish purple granules within cells that show up when stained with methylene blue
18
Q
Palisade Arrangement
A
-pertains to a parallel arrangement of rod-shaped cells
19
Q
Negative Stain
A
-cells appear as transparent objects against a dark background
20
Q
Capsular Stain
A
- all red background see white cells in clusters
- stain with CV for 2 minutes then wash off with aqueous solution of 20% copper sulfate and blotted dry
- under oil immersion capsules will appear as halos around cells and cells will be dark purple
21
Q
Spore Stain
A
- purple tapered rods
- schaeffer-fulton method: malachite green to stain spore and safranin to stain vegetative portion
- dorner method: produces a red spore within colorless sporangium. Nigrosin used to provide dark background for contrast
22
Q
Acid Fast Stain
A
- red with all white background
- important in identification of Mycobacterium tuberculosis and mycobacterium leprae
- Kinyoun acid-fast method: modification in shich the concentrations of primary stain, basic fuchsin, and phenol are increased making it unnecessary to heat cells during staining procedure
- increased concentrations of basic fuchsin and phenol are sufficient to allow the penetration of the stain into cells and the basic fuchsin is not removed during destaining with acid-alcohol
- acid-fast bacteria not decolorized by acid and are stained pink to red by fuchsin
- methylene blue is applied to see non-acid fast bacteria
23
Q
Gram Staining
A
- differential stain
- gram positive are purple and gram negative are red
- primary stain: crystal violet
- mordant: gram’s iodine
- decolorizer: alcohol
- counterstain: safranin
- gram positive cells have thick wall of peptidoglycan that is what forms a complex with the iodine to trap the crystal violet in the wall
24
Q
Aerobes
A
- found at top of tube
- respire O2
- FTM turns red at top
- cloudy at top in TYGA
- bacillus
25
Microaerophiles
- found 1/3 way down in tube
- require less O2
- FTM turns red a little bet below the top
- Bubbly/cloudy little below top in TYGA
26
Facultative
- found basically anywhere along tube
- respire O2 if present, ferment if not
- can't normally tell difference between these and aerotolerant but these respire at top and ferment at bottom and facultative always ferment at bottom
- FTM turns red at top and cloudy below where it ferments
- Bubbly throughout, cloudy at top for TYGA
- e. coli
- s. aureus
27
Aerotolerant
- ferment
- found at bottom of tube
- can't normally tell difference between these and facultative but facultatives respire at top and ferment at bottom and these always ferment at bottom
- FTM has no red but is cloudy low in tube
- bubbly througout
28
Anaerobes
- ferment
- found at bottom
- cloudy at bottom
- only bubbly well below surface
- clostridium
29
TYGA Shake Tube
- tryptone yeast glucose agar
- used to determine oxygen requirements for organisms
- inoculate as a liquid, roll between hands to mix then wait for it to solidify
- cracks happen when too much oxygen is produced
30
FTM Tubes
- fluid thioglycollate media
- used to determine oxygen requirements of organisms
- contain resazurin that turns red where oxygen is produced
31
GasPak Jar
- methylene blue strip
- anerobic jar
- used to prepare anaerobic organisms
32
Brewers Anaerobic Agar
-contains thioglycollate and resazurin and is plated and put in GasPak jar
33
Psychrophiles
-5-20 C
34
Psychrotrophs
-mesophiles that can grow in fridge
35
Mesophiles
- 20-50 C
| - most bacteria including human pathogens
36
Thermophiles
- 50-80 C
| - soil, compost piles
37
Hyperthermophiles
- >80 C
| - hot springs
38
Thermodurics
-survive but do not grow at high temps
39
Pigment Test
- inoculate 2 NA slants with Serratia and incubate in drawer and in incubator to determine best temp for pigment production
- 25 C is best
40
Growth Test
-helps determine best growth requirements for orgs by incubating them at several different temperatures
41
Neutrophiles
- grow best at neutral pH
| - staph and e. coli
42
Acidophiles
- grow best at acidic pH
| - saccharomyces cervisiae
43
Alkaliphiles
- grow best at basic pH
| - alcaligenes faecalis or sporosarcina ureae
44
pH Test
- test effects of pH on bacterial growth
| - determine best pH for some bacteria
45
Isotonic
-solute concentration same inside and outside cell
46
Hypotonic
-solute concentration higher inside cell, water moves into cell
47
Hypertonic
-solute concentration higher outside cell, water moves out of cell-->plasmolysis
48
Osmotic Pressure Test
- test 2 different bacteria at 3 salt concentrations to determine osmotic requirements
- halotolerant?
- e. coli and staph grew best at .5% NaCl
- halobacterium salinarium grew best at 15% NaCl
49
Osmophiles
-grow in environments where sugar concentrations are excessive
50
Halophiles
-require high concentrations of salt
51
Halotolerant
-capable of growth in moderate concentrations of salt
52
Defined Media
-know what's in it
53
Complex Media
- no idea of exact compounds present
| - allows growth of many types of organisms
54
Selective Media
- contains a growth inhibitor for specific group of organisms
- inhibitor is usually a dye, a specific chemical, NaCl, pH, or antibiotic
- organisms resistant to inhibitor will grow
- the dyes methylene blue and eosin in EMB media inhibit growth of gram positive bacteria
- incorporation of NaCl into mannitol-salt agar prevents bacteria that can't handle salt from growing
55
Differential Media
- usually contains pH indicator or in some way differentiates between physiological types of organisms
- mannitol-salt agar changes red to yellow around colonies that ferment mannitol
- EMB: gram negative bacteria that ferment lactose in this medium turn matallic green
56
Enriched Media
- compounds are added to further supplement the nutrients in complex media for fastidious organisms
- require certain nutrients (picky)
- ex: whole blood, serum, plasma, vitamins, and specific amino acids
57
Micropipettes
-the red handled guys with plunger
58
Pi-pumps
-the green and blue guys you put on end of glass pipette
59
Pour Plate
- inoculate bottom of empty petri dish
- pour melted agar over inoculum swirl in gentle figure 8's to mix
- get large surface colonies and small elliptical subsurface colonies
60
Serological Pipettes
-0-9 if liquid still left in tube you don't have all of what you measured
61
Measuring Pipette
-0-10 not lined all the way to the bottom of the tip
62
Volumetric Pipette
-has a bulb in it that holds liquid
63
Hockey Stick
- used for spread plates
| - glass
64
Optical Density
- measures turbidity related to growth
- construct a standard curve that relates optical density to actual numbers of bacteria determined by a SPC
- living and dead cells contribute to count
65
Standard Plate Count
- SPC
- serial dilution
- inoculate with e. coli and distribute dilutions to plates to be incubated and then counted later and then calculate cells per milliliter in undiluted culture
66
Determination of Growth by Absorbance
-serial dilutions that are then put through a spectrophotometer to measure absorbance which can be made into a curve to calculate maximum optical density
67
TGEA
- tryptone glucose extract agar
| - used for total counts of bacteria in hamburger
68
Bacterial Count in Hamburger
- inoculate 3 TGEA pour plates to check for total bacteria
- serial dilution
- incubate for 24 hours
- count colonies
69
MPN
-most probable number of coliforms present in water
70
Presumptive Test
- collect dirty water
- perform dilutions into test tubes with Durham tubes inverted inside
- both DSLB (double strength lactose broth) and SSLB (single strength lactose broth) tubes
- incubate for 24 hours and see if gas forms in durham tubes
- positive result if 10% or more gas is present in 24 hours presumed to be unsafe to drink
- determine MPN by using table and selecting three consecutive sets of tubes that have at least one tube with no gas
71
Confirmed Test
- gas accumulates in durham tubes so they're positive
| - choose one tube that has a positive result and do a streak plate onto EMB and Endo agar
72
Levine EMB Agar
- eosine methylene blue
- contains methylene blue which inhibits gram positve bacteria
- gram negative lactose fermenters that grow on this medium will produce nucleated colonies
- e. coli and e. aerogenes can be differentiated on basis of size and presence of green metallic sheen (e. coli are smaller and are green)
- selective and differential medium
73
Endo Agar
- contains fuchsin sulfite indicator that makes identification of lactose fermentors easy
- coliform colonies and surrounding medium appear red
- nonfermenters are colorless and don't affect color of medium
- some e. coli also turns metallic
74
Completed Test
- inoculating nutrient agar slant and lactose broth with Durham tube
- incubate 24 hours and lactose broth examined for gas production
- gram stain slide made and examined
- gram-negative non-spore forming rod that ferments lactose=coliforms present in water sample
75
Enterics
- gram negative, nonsporeforming, facultative anaerobic rods that ferment glucose with the production of acid and gas can grow in the intestinal tracts of warm-blooded mammals
- three groups: salmonella, shigella, and vibrio
76
Escherichia
- enteric
- number one cause of UTI in women
- septicemia-an infection of the blood stream
- spinal meningitis in newborns
- abdominal and wound infections
77
Salmonella
- enteric
- typhoid fever
- gastroenteritis
- osteomyelitis: infection in the bone
78
Shigella
- enteric
| - bacillary dysentery
79
Citrobacter
- enteric
| - abdominal and wound infections
80
Klebsiella
- enteric
- severe enteritis in children
- pneumonia and upper respiratory infections
- UTI of nosocomial (hospital acquired) origin
81
Enetrobacter
- enteric
| - abdominal and wound infections
82
Serratia
- enteric
| - thought to be innocuous, now implicated in pulmonary infections and septicemia
83
Proteus
- enteric
- number one cause of UTI in males
- abdominal and wound infections
84
MacConkey Agar
- selective due to bile salts and crystal violet and differential because it contains the pH indicator, neutral red
- tests for lactose fermentation and indicates that organism is a gram negative rod
- streak organisms for isolated colonies
- growth=gram negative rod
- positive=red colonies=lactose fermenter
- negative=colorless colonies=non-lactose fermenter
- all pathogens (salmonella and shigella) are lactose negative
85
TSI Agar
- Triple Sugar Iron Agar
- differentiates gram negative enteric bacteria
- stab loop down to center of butt all the way to bottom (inoculation for anaerobic growth)
- pull loop out of agar and zigzag streak up slant (inoculate for aerobic growth)
- incubate for 24 hours at 37 C
- time is critical after 24 hours tubes are unreliable
- TSI contains 3 sugars-10X more lactose and sucrose than glucose; sodium thiosulfate-detects organisms that can produce H2S and phenol red-alkaline Red=K and acid yellow=A
- first letter refers to top half and second refers to bottom stab (K/A A/A and K/K
86
Reactions of TSI Stabs
- K/A=only glucose fermented
- A/A=glucose and lactose or sucrose fermented
- K/K no sugar fermented (non fermentors)
- gas production is detected by gas bubbles in the agar
- so K/A + gas or A/A + gas
- some organisms produce H2x (salmonella and shigella) produces black precipitate so K/H2S or A/H2S
87
Urease
- splits urea into CO2 and ammonia
- ammonia cause pH to rise above 8.4 where phenol red, is a deeper red
- produced by some of the gram-negative enteric bacteria (proteus, providencia, morganella) which can be differentiated from the to others by this test
- inoculate urease test medium which is a light pink slant
- positive turns hot pink
- negative is light pink
88
Indole
- organisms that possess tryptophanase can cleave tryptophan to indole
- use Kovac's reagent to detect which produces cherry red color
- use indole test medium (tryptone broth which is pale yellow) and kovac's reagent
- inoculate broth and incubate at 37 C for 24-48 hours
- add 5 drops Kovac's reagent and wait
- positive=cherry red ring
- negative=yellowish ring
89
Citrate
- test medium contains ammonium phosphate which when utilized results in release of ammonium ions and an increase in pH.
- pH indicator Bromothymol Blue changes from green to blue above pH 7.8
- use simmons citrate slant which is green agar
- inoculate sland and incubate for 24 hours at 37 C
- positive=growth and slant turns blue
- negative=no growth
90
IMViC
- citrate and indole tests part of this
- used in water analysis
- Indole, Methyl red, Voges-Proskauer, Citrate
- Methyl red and Voges-Proskauer are tests of fermentation products
91
Non Fermenters
- aerobes
- Pseudomonas, alcaligenes, acinetobacter, and moraxella
- opportunistic pathogens
92
Pseudomonas
- causes UTIs, septicemia, upper and lower respiratory tract infections and colonizes wound and burn infections
- grown on light-colored media will produce a diffusible water soluble, green pigment called pyocyanin and a grape like odor
- oxidase positive
93
Acinetobacter
- isolated less frequently but can cause same type of infections but is less resistant to antibiotics
- Moraxella and alcaligenes are isolated even less frequently
94
Oxidase Test
- tests for presence of cytochrome C
- all organisms that can grow in the presence of air have cytochromes but they may differ
- oxidase reagent detects only C cytochrome
- thaw oxidase reagent
- wet very tip of q-tip
- touch tip to bacterial colony to be tested
- observe for color change and time reaction
- positive=turns dark purple in less than ten seconds
- negative=no color change or turns purple after twenty seconds
- questionable=turns purple at 10-20 seconds
95
API 20 E System
- mini version of conventional tests used for identification of members of the family enterobacteriaceae and other gram-negative bacteria
- utilizes plastic strip with 20 separate compartments each consisting of a depression/capsule and small tube that contains a specific dehydrated medium
96
Enterotube II
- mini multitest system developed for rapid identification of Enterobacteriaceae
- incorporates 12 different conventional media and 15 biochemical tests into a single ready to use tube that can be simultaneously inoculated
97
Host Specificity of T2 Bacteriophate
- determines if T2 bacteriophage is specific for E. coli or if it will also infect enterobacter aerogenes
- 4 tubes
- 500 microliters E coli in two tubes and then 500 microliters of T2 into on of those tubes
- 500 microliters of enterobacter into two new tubes and 500 microliters of T2 into one of those
- incubate at 37 C for 24 hours
- T2 only effectively kills E. coli
98
Quantitative Top Agar Assay of T2 Phage
- determines concentration of T2 bacteriophage in original broth using plaque count dilution assay
- 3 plates of bottom agar
- put pasteur pipette in e. coli tube
- add e. coli and T2 to small tube of top agar and mix well with finger flicks then pour over surface of bottom agar and rock quickly to spread
- plate 1: top agar and 2 drops e. coli
- plate 2: top agar and 2 drops e. coli and 100 microliters of T2
- plate 3: top agar and 2 drops of e. coli and 200 microliters of T2
- incubate at 37 C for 24 hours
99
Pasture Pipette
-very thin pipette with a bulb in middle
100
Bacteriophages
- obligate intracellular parasites
- have capsids that protects one type of nucleic acid
- can have a lytic or lysogenic life cycle
- after lysis they form clear areas called plaques
- PFU's can be counted to determine number of viral particles in a suspension of phage
101
Staphlococci
- means bunch of grapes
- gram positive
- non motile non spore forming and able to grow in high salt
- most are facultative anerobes
- s. aureus most important pathogen-salt tolerant, ferments mannitol, coagulase positive which means they cause a serum to clot
- DNase also associated with staph aureus as well as alpha toxin that causes wide, clear zone of beta-hemolysis on blood agar
102
MSA Plate
- mannitol salt agar
- production of acid by s. aureus that grows on this plate lowers the pH of the medium changing the phenol red indicator to turn yellow
- differential and selective
- differential because of mannitol (mannitol fermentors vs. non mannitol fermentors) and selective because of salt and selects for s. aureus because it survives in high salt
103
Coagulase Negative Staphylococci
- s. epidermidis and s. saprophyticus differ from s. aureus
- don't produce coagulase or DNase or alpha toxin
- all people have CNS on skin
- unpigmented and appear opaque when grown on blood agar and staphylococcus 110 plates
104
Beta-hemolysis
-complete lysis of red blood cells around a colony
105
Alpha-hemolysis
-partial breakdown of hemoglobin inside red blood cells on blood agar plate resulting in a greenish discoloration around colonies
106
Gamma-hemolysis
-no effect on blood cells in blood agar plate
107
Nose swab
- dip qtip into sterile water
| - swab nose and run on mannitol salt agar plate
108
Catalase Test
- place drop of H2O2 on microscope slide
- get glob of cells on loop (staph and strep)
- insert loop into drop
- vigorous bubbling=positive
109
Coagulase Test
- inoculate two tubes coagulase with staph aureus and staph epidermidis
- incubate 37 C for 24 hours
- thick is a positive result
110
Throat Swab
- used to look for hemolysis of strep
- swab throat and streak on blood agar plate
- examine after 24 hours
111
Thermal Death Point
- TDP
| - lowest temperature at which a population of a target organism is killed in 10 minutes
112
Thermal Death Time
-shortest time required to kill a suspension of cells or spores under defined conditions at a given temperature
113
Lethal Effects of Temperature
- plate control of culture
- place in water bath at constant temperature for 40 minutes
- every ten minutes plate .1 ml onto one of 4 plates and pour liquid nutrient agar into each plate rotate and cool
- incubate at 37 C for 24-48 hours
- results: strep faecalis is thermoduric; megaterium survives high temperatures because of endospores
114
Antimicrobials
-compounds that kill or inhibit microorganisms
115
Antibiotics
-are antimicrobials produced by microorganisms that inhibit or kill other microorganisms
116
Zone of Inhibition
-if agent kills or inhibits test organism this appears around the disc where no growth occurs
0can vary with diffusibility of agent and size of inoculum, type of medium etc
117
Antimicrobic Sensitivity Testing
- swab entire surface of TSA plate
- use dispenser to put down 12 discs
- tap down discs with sterile loop
- measure after 24 hours
- use chart to determine resistance, intermediate, or sensitive
118
Antiseptics
-substances that inhibit microbial growth or kill microorganisms and are gentle enough to be applied to living tissue
119
Disinfectants
-chemical agents that are applied to inanimate objects and are usually more harsh and damaging to living tissue
120
Sterilants/sporicides
-destroy all microbial life including endospores
121
Sanitizers
-reduce microbial numbers to safe level but don't completely eliminate all microbes
122
Evaluation of Antiseptics
- filter paper disc method
- TSA plate and inoculate with q tip all over surface
- dip steril disk halfway into agent
- place disk onto correctly labeled section of agar near the edge
- incubate 37 C for 24 hours then measure zone of inhibition
